Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to the use of strontium salts for obtaining pharmaceutical compositions intended for the prevention and treatment of osteoarthritis.
• strontium salts for therapeutic use has already been the subject of numerous publications and patents.
• US-A-4,152,431 describes alkali metal salts usable in the treatment of inflammation.
• WO-A-94/09798 discloses sulphate complexes of different metals, active in the treatment of skin diseases.
• the work of Olle Svensson et al. (Acta Path. Microbiol. Immunol. Scand., Sect. A, 93, (1985), 115-120) show that strontium plays a role in certain cases of rickets.
• strontium in the form of mineral or organic salts, has remarkable pharmacological properties and finds a very interesting therapeutic application in the prevention and treatment of osteoarthritis.
• strontium salts stimulate the synthesis, by human chondrocytes, of proteoglycans and type II collagen.
• strontium salts are therefore of great interest in the prevention and treatment of osteoarthritis. Strontium salts are therefore particularly effective during the repair process which occurs in the initial phase of the disease.
• the present invention relates to the use of bivalent strontium salts of organic or mineral acids for obtaining pharmaceutical compositions intended for the prevention and treatment of osteoarthritis.
• mineral acids used to salify strontium in order to obtain the salts according to the present invention there may be mentioned more particularly hydrochloric, sulfuric, nitric, carbonic and phosphoric acids.
• organic acids used to salify strontium in order to obtain the salts according to the present invention there may be mentioned more particularly tartaric, maleic, maleic, malonic, fumaric, gluconic, oxalic, lactic, succinic, methanesulphonic, ethanesulphonic, camphoric acids. , citric as well as 2- [N, N-di (carboxymethyl) amino] -3-cyano-4-carboxymethylthiophene-5-carboxylic acid.
• the preferred strontium salt of the present invention is the distrontic salt of 2- [N, N-di (carboxymethyl) amino] -3-cyano-4-carboxymethylthiophene-5-carboxylic acid which has been described in patent EP -B-0 415 850 for its anti-osteoporotic properties and its use in the treatment of skin and vascular aging, liver diseases and dental diseases.
• Osteoarthritis is characterized anatomically by an initial and primitive destruction of the articular cartilages.
• cartilage renewal is a very slow process consisting of a phase of resorption by the chondrocytes, directly compensated by a phase of formation by these same chondrocytes.
• cartilage renewal can accelerate, leading to an early cartilage repair reaction followed by cellular decompensation and degradation of cartilage.
• the repair reaction results from a clonal multiplication of the chondrocytes and from their increased synthesis of the matrix components of the cartilage (D. Hamerman et al., N. Eng. J. Med., (1989), 320 (20) , 1322- 1330).
• This homeostatic reaction is not adapted and depends on systemic hormones and growth factors whose secretions decrease with age.
• the resorption of cartilage is regulated by enzymes and free radicals produced by adjacent tissues, but also and above all by the chondrocyte itself.
• compositions will be presented in forms suitable for oral, parenteral, transcutaneous, nasal, rectal, perlingual administration, and in particular in the form of injections, tablets, sublingual tablets, glossettes, capsules, capsules, tablets, suppositories, creams, ointments, dermal gels, etc ...
• the pharmaceutical compositions according to the invention contain one or more excipients or vehicles chosen from diluents, lubricants, binders, disintegrating agents, absorbents, dyes, sweeteners, etc.
• the useful dosage varies according to the sex, age and weight of the patient, the route of administration, the nature of the condition and any associated treatments and ranges between 25 mg and 3 g of strontium per 24 hours, for example between 100 mg and 2 g of strontium per 24 hours.
• the chondrocytes are cultured for a short period in order to preserve their phenotype.
• cartilage is removed from the knees of human corpses immediately after death. We excise the surface layers and medians avoiding the calcified layer. The cartilage is cut into small fragments and then subjected to enzymatic digestion. The cartilage fragments are then successively treated with hyaluronidase, pronase and collagenase.
• the pieces of cartilage are incubated with hyaluronidase, previously dissolved in Dubbelco's Modified Eagle's medium (DMEM, ICN Biomedical) (0.5 mg / ml; 3 g of cartilage for 10 ml of enzymatic solution), for 30 minutes at 37 ° C and under constant agitation (200 revolutions per minute).
• DMEM Dubbelco's Modified Eagle's medium
• the cartilage fragments are placed in a pronase solution (1 mg / ml in DMEM; 3 g of cartilage per 10 ml of enzyme solution) and incubated with this enzyme for one hour at 37 ° C.
• the cartilage fragments are then incubated with collagenase (1 mg / ml; 3 g for 10 ml of enzymatic solution) dissolved in DMEM containing 1% of Ultrosser G (Gibco, Ghent, Belgium), for 20 hours at 37 ° C. and under constant agitation (200 revolutions per minute).
• the cells are filtered on nylon tulle (pore diameter: 25 ⁇ m), washed 3 times, counted (number varying from 1.10 6 to 5.10 6 cells / ml depending on the scope of the study) then resuspended in a medium appropriate culture.
• the cells are thus kept in suspension with constant stirring in a gyratory mixer (100 revolutions per minute) and under an atmosphere composed of 95% air and 5% carbon dioxide.
• the cells and the supernatant are separated by centrifugation (1000 revolutions per minute for 5 minutes).
• the cell aggregates and the supernatant are stored at -20 ° C.
• the chondrocytes ( ⁇ 1.5 ⁇ 10 6 cells / ml) are cultured for 24, 48 and 72 hours in the absence or in the presence of a strontium salt to be tested, at concentrations of 10 -4 M, 5.10 -4 M and 10 -3 M. For each concentration of compound to be tested and for the corresponding controls, a batch of three culture flasks was used. Each bottle contains ⁇ 1 to 1.5.10 6 chondrocytes. The experiment is repeated twice on two different donors. Proteoglycans and collagen II are measured in the supernatants and in the cell phases.
• ITS + contains: 6.25 ⁇ g / ml insulin, 625 ⁇ g / ml transferrin, 6.25 ⁇ g / ml selenium, 1.25 mg / ml bovine serum albumin and 535 ⁇ g linoleic acid.
• Proteoglycans and type II collagen are directly dosed in the supernatant previously added with protease inhibitors. Before the measurements, the cell aggregates are homogenized by ultrasonic dissociation (3 pulses of 10 seconds) in PBS containing the protease inhibitors.
• protease inhibitors used in this study are 6-aminohexanoic acid (0.1 M), benzamidine hydrochloride (0.05 M), a trypsin inhibitor (5.10 -8 M), EDTA (0, 01 M) and sodium nitride (6,7.10 -3 M).
• proteoglycans released into the culture medium (MC) and present in the chondrocytic aggregates (AC) are assayed according to the method described by P. Gysen and P. Franchimont (J. Immunoassay, (1984), 5, 221-243).
• the analytical sensitivity of the RIA is 0.6 ng / tubc.
• the intra- and inter-dosage coefficients of variation are less than 10 and 20% respectively, along the linear part of the curve.
• Antibodies are directed only to the antigenic determinant of the protein nucleus.
• the sum of the proteoglycans in the MC and in the AC corresponds to the total production of the molecule.
• This method assesses the production of sulfated proteoglycans by incorporation of 35 SO 4 .
• the chondrocytes treated with strontium salts are cultured in the presence of Na 2 35 SO 4 for 24 hours.
• the cells are separated for centrifugation and rinsed.
• the glycosaminoglycans present in the culture medium and the cells are extracted in the presence of protease inhibitors.
• the total production of sulfated glycosaminoglycans during the 24 hours of culture is evaluated by counting the radioactivity.
• the extracts are subjected to chromatography on CL 2B sepharose to study their physicochemical structure.
• the type II collagen released into the culture medium and present in the cell phase is assayed according to the RIA method described by Y. Henrotin et al. (J. Immunoassay, (1990), 11, 555-578).
• the detection limit for the assay is 3 ng / tube.
• the intra- and inter-dosage coefficients of variation are 8 and 15% respectively.
• the antibodies are directed on the helical part of the native molecule.
• TS + contains; 625 ⁇ g / ml transferrin, 6.25 ⁇ g / ml selenium, 1.25 mg / ml bovine serum albumin and 535 ⁇ g linoleic acid.
• Proteoglycans and type II collagen are directly dosed in the supernatant previously added with protease inhibitors.
• the cell aggregates are homogenized by ultrasonic dissociation (3 pulses of 10 seconds) in PBS containing the protease inhibitors.
• casein degradation activity is assayed by labeled casein-resorufin from cow's milk (Boehringer Mannheim).
• the activation of latent stromelysin is produced by the addition of APMA (para-aminophenylmercuric acetate, Sigma Chemie, Deinsenhofen, Germany) at the final concentration of 2 mM.
• APMA para-aminophenylmercuric acetate
• the conditioned medium of the chondrocytes is incubated for 4 hours at 37 ° C.
• the enzymatic reaction is stopped by adding trichloroacetic acid to the final concentration of 1.6%.
• the sample is centrifuged at 7000 g for 15 minutes. Under these conditions, the uncleaved casein is completely precipitated, while more than 95% of the cleaved casein remains in the supernatant.
• the measurements are carried out by fluorescence.
• the excitation wavelength is 574 nm and the emission wavelength is 584 nm.
• the standard curve is obtained by incubation of increasing amounts of purified stromelysin.
• samples containing 10 mM EDTA added before incubation are treated in a similar manner, providing control values which are subtracted from the values obtained with the active medium.
• the control values never exceed 5% of the fluorescence measured in the active medium. All assays are performed in duplicate.
• results are expressed in ratios of proteoglycans and type II collagen released in the culture media and present in the cell phases, per ⁇ g of DNA.
• the means ⁇ the standard deviations of the means are calculated. Comparisons between groups are made using the unpaired Student's t-test; differences are considered to be statistically significant when p ⁇ 0.05.
• the chromatographic profiles obtained with the two strontium salts and in particular with compound A demonstrate an increased synthesis of high molecular weight glycosaminoglycans.
• the table below presents the distribution (%) of the proteoglycans synthesized as a function of their molecular weight.

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• Physical Education & Sports Medicine (AREA)
• Inorganic Chemistry (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Rheumatology (AREA)
• Immunology (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
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• 1996
  • 1996-06-17 FR FR9607475A patent/FR2749759B1/fr not_active Expired - Fee Related
• 1997
  • 1997-05-30 CA CA002206837A patent/CA2206837C/fr not_active Expired - Fee Related
  • 1997-06-05 JP JP14747497A patent/JP3935557B2/ja not_active Expired - Fee Related
  • 1997-06-11 US US08/873,117 patent/US5856356A/en not_active Expired - Lifetime
  • 1997-06-13 AU AU24902/97A patent/AU711950B2/en not_active Ceased
  • 1997-06-16 DK DK97401362T patent/DK0813869T3/da active
  • 1997-06-16 AT AT97401362T patent/ATE233558T1/de active
  • 1997-06-16 ES ES97401362T patent/ES2193337T3/es not_active Expired - Lifetime
  • 1997-06-16 CN CNB971149089A patent/CN1136856C/zh not_active Expired - Fee Related
  • 1997-06-16 NO NO19972764A patent/NO315840B1/no not_active IP Right Cessation
  • 1997-06-16 DE DE69719408T patent/DE69719408T2/de not_active Expired - Lifetime
  • 1997-06-16 NZ NZ328106A patent/NZ328106A/en not_active IP Right Cessation
  • 1997-06-16 EP EP97401362A patent/EP0813869B1/de not_active Expired - Lifetime
  • 1997-06-17 BR BR9703603A patent/BR9703603A/pt not_active IP Right Cessation
  • 1997-06-17 ZA ZA9705323A patent/ZA975323B/xx unknown
  • 1997-06-17 HU HU9701059A patent/HU227935B1/hu not_active IP Right Cessation

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